The global impact of smoking on public health remains devastating. Despite aggressive efforts to reduce cigarette consumption, smoking-related lung diseases such as COPD and lung cancer continue to dominate health care headlines. Even with successful smoking cessation, the risk of developing lung disease persists for years and perhaps decades. Passive or environmental smoke exposure increases the risk for certain lung disorders suggesting that there may be no safe threshold for smoke inhalation. How smoking results in an enduring disease risk with low or no threshold is not currently understood. The resident alveolar macrophage has been implicated in the pathophysiology of many smoking-related lung diseases. Preliminary work in our laboratory has uncovered significant changes in both microRNA expression and methylation status of CpG motifs in the promoter regions in the alveolar macrophages from active smokers compared to never smokers. This proposal will investigate the overall hypothesis that in vivo cigarette smoke exposure alters the genomic tone of alveolar macrophages contributing to the development of smoking-related lung diseases. The focus for this project is microRNA expression profiles and gene promoter DNA methylation status as two critical regulatory points in cellular gene expression. In Aim 1, we will examine the hypothesis that chronic cigarette smoke exposure causes a global down regulation of microRNA expression in alveolar macrophages by interfering with the microRNA maturation process. To explore the threshold needed to produce this response, we will study microRNA profiles in alveolar macrophages obtained from domestic partners of active smokers; to define the persistence of gene suppression we will study cells from ex-smokers. In Aim 2, we will examine the hypothesis that active smoking alters DNA methylation patterns in gene promoter regions by comparing DNA CpG methylation profiles. We will study the same active smokers, ex-smokers and individuals with passive smoke exposure as in Aim 1. Finally, Aim 3 will validate the array findings (microRNA, mRNA and DNA methylation) with targeted individual assays. Differentially expressed microRNAs and altered methylated CpG islands in promoter regions will be correlated with exon-specific mRNA expression via bioinformatics analysis. In the case of microRNAs, over and under expression studies will confirm links between altered microRNAs and potential targets. Identification of a specific genomic phenotype in the alveolar macrophage from smoke-exposed subjects would provide important insight into pathogenesis of smoking-related lung disease. In addition, this could lead to an easily retrievable biomarker useful in tracking physiologically relevant smoke exposure and may be applicable to lung disease related to other inhaled toxicants.